Extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge because formaldehyde, the effective component of formalin, leads to the generation of cross-linking between nucleic acids and proteins, and additionally causes nucleic acids to fragment because of fixation process conditions, e.g., the extremely low pH (Lin et al., Anal Biochem., 395(2): 265-267 (2009). Furthermore, the detection of circulating cell free DNA (cfDNA) and RNA in human blood, which has generated a lot of interest for its use in the so called liquid biopsy approach, has certain limitations, including the fragmented nature of such DNA with the average size being about 160 to 180 bp (Suzuki et al., Clinica Chimica Acta, 387, 55-58 (2008)).
For PCR based cancer diagnosis assays, the size limitation for fragmented DNA may not be a problem for mutation hot spots like BRAF V600, KRAS G12/G13, or PIK3CA E542/E545/E546 where the mutations duster in few nearby codons, but it is a challenge for others like EGFR exon 20 where mutations spread over 25 codons. The abundance of intact DNA in any sample may also be insufficient for accurate and sensitive detection of multiple mutation sites, so a method of target pre-enrichment before mutation detection is desirable. Methods for the pre-enrichment of target Nucleic Acids are known, but these methods involve multiple steps of reagent addition or are methods for the linear amplification of the target strands. The present disclosure addresses methods of such target pre-enrichment in an exponential and homogeneous manner without the need for multiple discrete steps and reagents. Therefore a method that can bring overlapping short nucleic acid fragments together and thus serve as the template for downstream PCR detection is desirable, which the present disclosure addresses.